THE  ASYMMETRY  OF  THE  ALIPHATIC 
DIAZO  ESTERS 


BY 

HOWARD  MARION  CHILES 

B.S.  University  of  Illinois,  1917 
M.S.  University  of  Illinois,  1920 


THESIS 

SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS 
FOR  THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY  IN  CHEMISTRY 
IN  THE  GRADUATE  SCHOOL  OF  THE  UNIVERSITY 
OF  ILLINOIS.  1922 


URBANA,  ILLINOIS 


&.2L. 


Q-Mj 


Digitized  by  the  Internet  Archive 
in  2015 


https://archive.org/details/asymmetryofaliphOOchil 


ACKNOWLEDGMENT 


The  writer  wishes  to  express  his  thanks 
to  Professor  W.  A.  Noyes  for  his  assistance 
and  interest  in  this  investigation,  which  was 
proposed  by  him  and  carried  out  under  his  dir- 
ection. He  also  wishes  to  thank  Dr.  C.  S. 
Marvel  for  mary  helpful  suggestions  which  he 
has  given  as  well  as  for  a part  of  the  mater- 
ials used  which  were  prepared  by  him. 


I INTRODUCTION 


1 

A little  more  than  a year  ago  Levene  and  Mikeska  publish- 
ed a paper  in  which  they  give  experimental  evidence  for  the  as- 
symetry  of  diazo  diethyl  succinate*  This  is  the  first  direct 
evidence  which  has  been  produced  of  the  assymetry  of  an  alipha- 

3 

tic  diazo  compound.  The  work  of  Noyes  and  Marvel  produced  in- 
direct evidence  of  the  existence  of  such  compounds  by  the  pro- 
duction of  slightly  active  hydroxy  esters  by  the  treatment  of 
amino  ester  hydrochlorides  with  nitrous  acid  in  such  a way  that 
the  diazo  compounds  were  decomposed  without  being  isolated. 

The  present  work  on  aliphatic  dizao  compounds  was  started 
before  the  appearance  of  the  paper  of  Levene  and  Mikeska  and  it 
was  thought  desirable  to  continue  the  work  on  the  aliphatic 
diazo  compounds  not  only  to  verify  their  work,  but  also  to  pro- 
duce other  similar  compounds  and  to  produce  further  evidence  to 
show  that  the  activity  observed  was  actually  due  to  a difference 
in  the  nitrogen  atoms  in  the  molecule  and  not  to  some  other 
cause. 

According  to  the  Curtius  formula  for  the  aliphatic  diazo 
compounds  two  nitrogen  atoms  are  attached  to  the  same  carbon 
atom,  while  from  the  method  of  formation  of  the  diazo  compounds 
one  of  the  nitrogen  atoms  comes  from  ammonia  while  the  other 
comes  from  nitrous  acid.  These  compounds  have  the  following 
electronic  formulas:- 


*“t_  * 


iT< 


.H+ 

H+ 

'H+ 


+ — +++  — 
H - 0 - N = 0 


The  electronis  formula  for  an  aliphatic  diazo  compound 
could  then  be  written  according  to  the  Curtius  formula. 

r^v*; 

According  to  the  Angeli-Thiele  formula  for  the  aliphatic 
diazo  compounds  which  has  recently  won  a large  number  of  new 
adherents,  the  electronic  formula  could  be  written:- 

R\- 

\C+  = N 2 N 
BT 

It  can  be  seen  that  even  with  this  formula  a possibility 
of  assymetry  exists. 

The  first  aliphatic  diazo  compound,  diazo  camphor,  was  pre- 

3 

pared  by  Schiff  and  Meissen  by  the  action  of  nitrous  acid  on 
camphor-imide . It  was  Curtius  however  who  first  made  a diazo 

4 

compound  from  an  amino  ester  by  the  action  of  nitrous  acid  on 

glycocoll  ester  hydrochloride.  He  made  a thorough  study  of  the 

reactions  of  this  compound  as  well  as  a number  of  other  alipha- 

8 

tic  diazo  compounds  and  assigned  the  structure 

R Vc\~  R 

if  = U 

The  principal  reactions  on  wnich  the  structure  of  the  aliphatic 

diazo  compounds  are  based  are  as  follows:-  With  iodine  ethyl 

diazo  acetate  gives  di-iodo  ethyl  acetate  upon  reduction  with 

e 

zinc  and  acetic  acid  the  original  amino  ester  is  obtained. 


-3- 


When  reduced  with  sodium  amalgam  the  hydrazinoacetate  is  ob- 

7 

tained  • With  concentrated  hydrochloric  acid  it  gives  ethyl 

monochloroacetate.  By  the  action  of  water  or  dilute  acids  the 

corresponding  hydroxy  ester  is  obtained.  By  the  action  of  an 

acyl  chloride  on  two  moles  of  ethyl  diazo  acetate,  one  mole  of 

ethyl  monochloroacetate  is  formed  and  one  mole  of  the  acyl 

diazo  ester  8 

R~C  - C - C-0— R. 

Ha 

By  the  reduction  of  ethyl  diazo  acetate  with  palladium  black 
o 

and  hydrogen  the  hydrazone  is  obtained. 

It  was  apparently  this  last  bit  of  evidence  which  caused 

Staudinger,  so  long  an  advocate  of  the  Curtius  formula,  to  lend 

10 

his  support  to  the  Thiele-Angeli  formula,  for  in  1916  after 
a complete  study  of  the  reactions  of  these  compounds  admitted 
that  Curtius*  ring  formation  still  explains  matters  best  if  it 
it  assumed  that  the  ring  can  easily  be  ruptured  under  appropri- 
ate treatment.  In  the  same  paper  he  states  that  the  introduc- 
tion of  one  or  two  carbonyl  groups  at  the  methylene  carbon  of 
a diazo  compound  diminishes  the  color  and  lowers  the  reactivity 
of  the  molecule,  wnile  in  other  cases  the  introduction  of  a 
carboxyl  adjacent  to  unsaturated  groupings  enhances  the  color 
and  reactivity. 

In  this  series  of  work  Staudinger  and  his  co-workers  tried 
without  success,  to  isolate  isomeric  diazo  compounds,  of  the 

l l 

two  structures  R - C - R and  R C - R 


N = H 


R 

ui 

N 


■ 

. 


.....  . ; 

■ , i 


%< 


; f ......  , 


. 

" 

: ; - ‘i 

. . . . ' ■ . 

. 

* 

i 

...  • • 

t 

• 

. 

■.  . - £■ 

V 

■ 

- 

: 

- 

. 

-• 

” 

-4— 


II  THEORETICAL 


Methods  of  Preparation  j2£  the  Aliphatic  Diazo  Compounds. 

The  most  gexieral  method  for  the  preparation  of  the  alipha- 
tic  diazo  compounds  is  that  of  Curtius  toy  the  action  of  nitrous 
acid  on amino  ester  hydrochlorides.  This  method  nas  been  ex- 
tended so  as  to  include  diazo  compounds  prepared  by  the  action 
of  nitrous  acid  on  other  amino  derivatives,  such  as  cyanides, 
ketones,  imides  and  amides.  A general  method  for  their  prepara— 

13 

tion  is  that  of  v.  Pechmann  and  a modification  of  this  method 

1 3 

introduced  by  Oppe  by  which  the  diazo  compounds,  are  produced 
by  the  action  of  concentrated  alcoholic  or  aqueous  alkali  or  an  j 
etheral  solution  of  sodium  alkoxides  on  nitroso  imides.  The 
other  general  method  for  the  preparation  of  tne  aliphatic  diazo 
compounds,  that  of  oxidation  of  hydrazones  with  yellow  mercuric 

14 

oxide  is  not  applicable  for  the  preparation  of  optically 
active  diazo  compounds  due  to  the  fact  that  trie  hydrazones  are 
symmetrical  in  structure. 

The  only  pure  diazo  esters  prepared  in  this  work  were  ob- 
tained by  the  Curtius  method  and  purified  by  vacuum  distillation 
of  the  thoroughly  washed  and  dried  crude  samples. 

The  following  diazo  esters  were  prepared:- 

Impure  ethyl  and  methyl  esters  of  Y diazo  valeric  acid  were 
prepared  by  the  treatment  of  the  lactam  of  Y amino  valeric  acid 
with  sodium  ethylate  and  methylate.  No  method  of  purification 
could  be  found  due  to  the  extreme  instability  of  the  products. 

Diethyl^ iazoglutarate  was  prepared  by  the  action  of 


-5- 


nitrous  acid  d-diethyl  glutamate  hydrochloride  according  to  the 

Curtius  method.  The  purified  product  from  different  prepara- 

o o 

tions  had  a specific  rotation  of  + 0,85  to  + 1*68  * 

Dimethyl  <=<diazoglutarate  was  prepared  in  the  same  manner 

from  the  corresponding  dimethyl  ester  hydrochloride*  This  sub- 

o 

•stance  had  a specific  rotation  of  + 0,89  * 

Diethyl  «=<  diazo  succinate  was  prepared  in  a similar  manner 

from  1-diethyl  asparate  hydrochloride.  The  purified  product 

o 

had  a specific  rotation  of  - 1.23  . 

Ethyl^diazo  n-caproate  was  prepared  in  the  same  way  from 

l-ethjrl  «=><amino-n-caproate  hydrocriloride  and  had  a specific 

0 0 

rotation  of  - 1,89  • 

Ethyl  o<  diazo-n-caproat e was  prepared  from  the  d-ethyl 

0 

ester  hydrochloride  and  had  a specific  rotation  of  + 1,84  , 

Ethyl  diazo  isocaproate  was  prepared  from  the  levorota- 

tory  amino  ester  hydrochloride  and  had  a specific  rotation  of 
0 

- 1,53  * 

These  diazo  esters  upon  treatment  with  dilute  sulphuric 
acid  all  gave  a mixture  of  an  unsaturated  hydroxy  with  the  same 
cirection  of  rotation  as  the  amino  esters  and  diazo  esters  from 
which  these  were  prepared,  and  of  the  same  order  of  magnitude  as; 
the  diazo  ester. 

The  mixture  of  d-hydroxy  glutaric  esters  and  unsaturated 
ester  upon  saponification  gave  sodium  salts  with  the  same  direc- 
tion of  rotation  as  the  amino  acid,  hut  the  rotation  correspond-' 

ed  to  only  about  10--13  per  cent  of  the  pure  active  sodium  salt, 

products 

The  acid  hydrolysis/from  the  other  diazo  compounds  upon  sap-j 


■- 

- 

. 


-6- 


onif icat ion  gave  sodium  salts  which  had  the  same  direction  of 
rotation  but  in  some  cases  the  salts  were  not  identified  due  to 
lack  of  material. 

Diethyl^iazoglutarat  e upon  reduction  gave  d-diethyl  gluta-i 
mate  but  the  activity  corresponded  to  only  about  12  per  cent  of 
the  pure  active  amino  ester. 

The  rotation  of  the  diazo  glutaric  esters  prepared  cannot 
be  due  to  the  presence  of  the  amino  ester  as  this  product  was 
specifically  tested  for  by  the  Van  Slyke  nitrous  acid  method, 
after  treatment  with  dilute  acid  to  destroy  the  diazo  ester,  and! 
the  total  amount  of  nitrogen  obtained  calculated  as  the  amino 
ester  the  amounts  obtained  were  negligable,  and  moreover  the 
amino  ester  gives  only  a normal  increase  in  rotation  when  added 
to  the  diazo  ester.  The  rotation  can  not  be  due  to  the  presence 
of  a hydroxy  ester  as  the  rotations  of  the  hydroxy  ester  prepar- 
ed by  treatment  of  the  amino  ester  with  nitrous  acid  so  as  to 
decompose  the  diazo  esters  without  isolation,  were  of  the  same 
order  as  those  of  the  diazo  esters.  This  of  course  might  sug- 
gest that  the  same  impurity  caused  the  rotation  in  the  two  cases> 
but  it  is  hard  to  see  how,  even  if  this  supposed  impurity  escap- 
ed removal  by  distillation,  it  should  give  both  an  active  hy- 
droxy acid  upon  saponification  and  an  active  amino  acid  upon 
reduction,  and  that  the  rotation  obtained  although  of  a different 
amount  corresponds  at  least  approximately  to  the  same  percentage 
of  the  active  compound  in  the  two  cases. 

The  rotation  cannot  be  due  to  the  hydroxy  acid  as  this  com- 


■ 

. 

. 

. 

• v>  t 

V • 

• 

...  . 


, 


: 


‘ 


» 


. 


-7- 


15,  le 

pound,  in  the  first  place  is  levorotatory  , and  moreover  it 

certainly  would  be  separated  by  distillation.  The  rotation 
cannot  be  due  to  the  presence  of  pyrrolidone  carooxylic  ester, 

17 

as  this  compound  besides  being  levorotatory  has  a much  higher 
boiling  point. 

The  arguments  which  have  been  advanced  in  the  case  of  the 
glutaric  esters  in  the  main  hold  good  for  all  of  the  other  diazo 
esters  prepared  as  well,  though  in  some  cases  the  diazo  com- 
pounds prepared  were  not  as  pure  as  was  obtained  in  the  case  of 
the  glutaric  esters,  it  was  found  in  all  cases  that  purification 

had  onljr  a small  effect  on  the  rotation,  presumedly  due  to  the 

active 

fact,  that  the  chief/ impurity,  the  hydroxy  esters  in  all  cases 
had  rotations  of  the  same  order  as  that  of  the  corresponding 
diazo  compounds. 

It  has  been  rather  a common  observation  tha'D  in  the  distil- 
lation of  diazo  esters  even  those  that  have  been  carefully  dried 

a part  and  in  some  cases  nearly  all  of  the  diazo  ester  is  decora- 

18  , 18 

posed  with  the  production  of  a large  amount  of  hydroxy  ester 
It  was  found  in  tnis  work  that  when  the  diazo  esters  were  treat- 
ed with  metallic  sodium  in  ether  or  petroleum  ether  solution 
that  the  ether  solution  became  inactive  long  before  all  of  the 
diazo  ester  was  destroyed.  When  the  diazo  ester  is  successively 
treated  with  sodium  methylate  in  ether  solution  and  distilled 
for  from  4 to  6 times  the  diazo  ester  finally  becomes  entirely 
inactive.  When  the  diazo  e3ter  is  reduced  only  approximately 
12  per  cent  of  the  glutamic  acid  obtained  is  active,  and  approx- 


-3- 


imately  the  same  percentage  of  active  hydroxy  acid  is  obtained 
by  treatment  with  acid  followed  by  saponif ication  of  the  hydroxy 
ester.  Moreover  we  nave  never  obtained  diazo  esters  showing 
over  99  per  cent  of  the  theoretical  amount  of  nitrogen  by  ana- 
lysis. These  results  might  be  taken  as  evidence  that  the  diazo 
esters  as  ordinarily  prepared  and  dried  contained  some  and  in  a 
few  cases  quite  a large  amount  of  the  hydrated  form,  which  might 
be  represented  by  the  formula 

R - § R1 

\l— N -OH 

However,  it  would  not  appear  likely  that  such  a compound  would 
distill  without  entire  decomposition,  in  fact  it  is  usually  as- 
sumed that  this  type  of  compound  is  formed  intermediately  in  the 
formation  of  diazo  esters,  and  then  it  is  immediately  and  com- 
pletely dehydrated.  Attempts  were  made  in  this  work  to  isolate 

the  benzoyl  derivatives  of  the  diethyl  diazoglutarate,  accord- 

20 

ing  to  the  method  used  by  Staudinger  in  the  case  of  diazo 
acetic  ester.  It  is  readily  seen  that  a compound  of  this  type 
would  not  be  formed  from  diazoglutaric  esters  according  to  the 
Curtius  formula.  The  attempts  to  isolate  such  a compound  was 
unsuccessful,  although  the  mixture  of  diazo  ester  and  benzoyl 
bromide  retained  the  diazo  color  after  the  benzoyl  bromide  odor 
had  disappeared.  The  bromoester  was  isolated  by  vacuum  distil- 
lation of  the  reaction  mixture,  but  neither  the  residue  from 
the  distillation  of  the  bromoester  or  the  original  reaction  mix- 
ture evolved  nitrogen  either  in  the  cold  or  when  heated  with 
twenty  per  cent  sulphuric  acid. 


-9- 


It  has  long  been  known  that  optically  active  hydroxy  acids 
may  be  obtained  by  the  treatment  of  amino  acids  with  nitrous 

21,32,23,24,25 

acid  * Diazo  esters  may  be  obtained  from  almost 

any  of  the  amino  esters  and  these  give  hydroxy  esters  when 
treated  with  water  or  dilute  acids*  Optically  active  hydroxy 
esters  have  been  prepared  by  the  action  of  nitrous  acid  in  such 

26,2  7 

a.  way  that  the  diazo  compound  may  be  an  intermediate  product  • 

28 

These  facts  are  considered  by  some  as  evidence  that  diazo  com- 
pounds are  intermediate  products  in  the  formation  of  hydroxy 
acids  when  amino  acids  are  treated  with  nitrous  acid.  However, 
it  should  be  noted  that  in  most  cases  the  hydroxy  acid  obtained 
rotates  in  the  opposite  direction  to  that  of  the  amino  acid 
from  which  it  was  prepared,  and  at  least  in  most  cases  in  which 
hydroxy  acids  rotate  in  the  same  direction  as  the  amino  acids, 
the  direction  of  rotation  of  the  amino  acid  itself  is  in  doubt 
as  the  direction  of  rotation  may  be  changed  by  choice  of  solvent 
or  the  formation  of  a salt  with  the  amino  or  carboxyl  groups. 

In  this  work  it  was  found  in  each  case  that  the  amino  esters 
gave  hydroxy  esters  with  the  same  direction  of  rotation  as  the 
amino  esters  from  which  prepared,  and  the  hydroxy  esters  in  turn 
gave  hydroxy  acids  with  the  same  direction  of  rotation.  Levene 

29 

and  Mikeska  found  that  dextro  diethyl  asparate  gave  the  d- 
hydroxy  ester  going  through  the  diazo  ester.  While  it  is  known 

30 

that  1 -aspartic  acid  gives  the  1-hydroxy  acid  . She  aspartic 

acid  itself  is  strongly  dextrorotatory  in  a decidedly  acid  sol- 
ution. These  facts  makes  it  appear  unlikely  that  diazo  compound:; 

are  intermediate  products  in  the  conversion  of  free  amino  acids 
into  the  hydroxy  acids  by  treatment  with  nitrous  acid. 


-10- 


III  EXPERIMENTAL 

The  first  attempt  made  was  the  preparation  of  ethyl/diazo- 
valerate,  by  the  treatment  of  nitroso  methyl  2 pyrrolidone  with 
an  etheral  solution  of  sodium  methylate  according  to  Oppe's 
method.  By  this  method  it  is  believed  both  the  diazo  ester  and 
sodium  diazo  oxide  were  prepared  but  neither  could  be  obtained 
in  a pure  condition,  owing  to  the  extreme  instability  of  the 
products. 

Preparation  of  / Amino  Valeric  Acid. 

Y Amino  valeric  acid  was  prepared  by  the  aluminium  amalgam 
reduction  of  the  phenylhydrazone  of  levulinic  acid  according  to 

3 1 

the  method  of  Fischer  and  Groh  . Using  116  grams  of  levulinic 
acid  dissolved  in  750  cc.  of  water  and  adding  this  with  effi- 
cient mechanical  stirring  to  a solution  of  108  grams  of  phenyl 
hydrazine  dissolved  in  750  cc.  of  water  with  just  enough  acetic 
acid  added  to  dissolve  the  phenyl  hydrazine.  The  yield  of  pink-' 
ish  yellow  crude  phenyl  hydrazone  was  200  grams  and  was  used  foi 
the  reduction  without  purification. 

The  reduction  was  carried  on  using  mechanical  stirring, and 
from  200  grams  of  the  crude  phenyl  hydrazone  we  obtained  in  dif- 
ferent runs  from  60-70  grams  of  crude  gamma  amino  valeric  acid, 

o 

melting  at  192-194  . Some  difficulty  was  found  in  obtaining 
suitable  aluminium  for  the  reduction.  None  of  three  different 
samples  of  granulated  aluminium  of  domestic  manufacture  would 


■ 


' 

■ 

a — 


. 


- 


•> 

, 


. 


-11- 

react  longer  than  a few  minutes  after  amalgamation*  Kahlbaum' b 

% 

aluminium  reacted  properly  as  did  aluminium  turnings  made  from 
aluminium  bars  and  finely  cut  aluminium  sheets  of  domestic  man- 
ufacture* 

Preparation  of  Nitroso  Methyl  2 Pyrrolidone 

This  compound  was  prepared  bjr  the  action  of  nitrous  acid 
upon  the  aqueous  solution  of  the  lactam  of  gamma  amino  valeric 

33 

acid  according  to  the  method  of  Tafel  • From  20  grams  of  gamma 
amino  valeric  acid  we  obtained  13  »1  g of  the  lactam  boiling 
138-140°  22  mm.  77  per  cent  of  the  theoretical  amount.  From 
this  we  obtained  6 grams  of  nitroso  methyl  2 pyrrolidone  as  a 
yellow  oil  which  could  not  be  crystallized.  The  product  gave 
Libermann's  nitroso  reaction.  The  product  was  analyzed  by 
treating  with  10  per  cent  sodium  hydroxide  and  measuring  the 
evolved  nitrogen. 

Analysis:  .0889  grams  of  substance  gave  13*05  cc.  nitrogen 
at  27*5°  and  747  mm. 

Attempts  to  Prepare  Diazo  Ethyl  Valerate  and  Diazo 

Methvl  Valerate. 

5.  g of  nitroso  methyl  2 pyrrolidone  prepared  as  just  des- 
cribed was  dissolved  in  3°0  cc.  of  very  carefully  dried  ether. 

o _ 

The  solution  was  cooled  to  -15  to  -20  and  a solution  of  .8  g 
of  sodium  in  10  cc.  of  absolute  ethyl  alcohol  was  added  in  small 
portions.  The  ether  solution  became  colored  a reddish  yellow. 

After  thirty  minutes  carefully  dried  carbon  dioxide  was  passed 
into  the  solution,  whereupon  the  ether  solution  almost  complete- 


-12- 


ly  lost  its  color.  After  filtration  in  dry  air  the  ether  solu- 
tion was  concentrated  by  evaporation  in  a vacuum  dessicator.  A 
small  amount  of  a sharp  smelling  liquid  of  a slightly  orange 
yellow  color  was  left.  The  product  would  not  solidify  upon 
cooling  with  freezing  mixture.  When  treated  with  dilute  acids 
there  was  a small  evolution  of  nitrogen.  The  product  upon 
vacuum  distillation  was  a colorless  liquid. 

A number  of  other  attempts  were  made  by  varying  the  amounts 
of  sodium  and  alcohol  used,  and  also  by  the  use  of  sodium  in 
methyl  alcohol,  but  in  all  cases  the  large  part  of  the  product 
was  decomposed  upon  the  addition  of  the  carbon  dioxide. 

Probably  the  best  yield  of  the  diazo  compound  was  formed 
by  the  use  of  about  *1  g sodium  methylate  in  a rather  large 
excess  of  methyl  alcohol.  In  this  case  the  ether  solution  was 
colored  a very  deep  cherry  red.  A part  of  this  solution  was 
treated  with  carbon  dioxide  in  the  usual  way  with  same  results 
as  above,  the  other  portion  of  the  solution  was  concentrated 
to  a small  volume  in  vacuum  dessicator,  but  it  was  impossible 
to  obtain  ary  separation*  The  addition  of  water  caused  an  evolu-** 
tion  of  nitrogen. 

Attempts  were  also  made  by  using  just  slightly  less  than 
the  theoretical  amounts  of  sodium  methylate  or  ethylate  to  ob- 
tain the  pure  sodium  diazo  oxide  compound  but  in  all  cases  a 
mixture  was  obtained. 

O.O569  of  the  purest  material  obtained  gave  6.1  cc  of  nitro- 
c 

gen  at  24  and  743  mm.  when  treated  with  dilute  sulphuric  acid. 


-13- 


The  sulphuric  acid  solution  was  evaporated  to  dryness  and  ignit- 
ed and  gave  *0272  g of  sodium  sulphate.  This  corresponds  rough- 
ly to  two  moles  of  sodium  to  one  of  diazo.  From  the  nature  of 
the  compound  which  was  a light  yellow  amorphous  solid,  quite  siiQ" 
ilar  to  the  product  obtained  when  a diazo  ester  is  treated  with 
sodium  methylate,  it  probably  is  a compound  of  the  type  of  the 
potassium^  diazo  acetate  mixed  with  sodium  ethylate,  which  has 
been  prepared  from  diazo  acetic  ester  by  treatment  with  potas-  j 

33,34 

sium  ethylate  . This  compound  was  decomposed  when  treated 

with  dilute  acids  or  water,  it  gave  a turbid  solution  in  abso- 
lute alcohol  which  could  not  be  clarified  by  filtering,  and  was 
either  decomposed  or  insoluble  in  all  of  the  common  organic 
solvents.  As  no  method  could  be  found  of  purifying  the  diazo 
compounds  obtained  the  work  on  these  compounds  was  discontinued. 

d-Glutamie  acid  was  chosen  as  the  starting  material  in  a 
large  part  of  the  work  because  from  its  ester  hydrochloride  the 
diazo  ester  can  be  obtained  in  fair  yields.  The  active  amino 
acid  can  be  obtained  in  a high  state  of  purity  directly  and 
with  comparative  ease,  and  further  it  was  thought  advisable,  in  1 
view  of  the  results  of  Noyes  and  Marvel,  to  prepare  the  diazo 
ester  of  a dibasic  acid  in  hopes  that  the  results  might  be  more 
positive  than  in  the  case  of  the  monobasic  acid  esters  prepared 
by  them. 

In  most  cases  the  amino  ester  hydrochloride  was  made  direct- 
ly from  the  acid  hydrochloride  and  the  diazo  ester  made  from 
the  recrystallized  amino  ester  hydrochloride.  Trial'  runs  were 


i 


- 

■ *,  - • 

• 

: 

. 

. 

. 

I 

■ 

■ 

-s 

•< 

1 

• 

. 

* 

. 

* 

• 

-14- 


made,  however,  in  which  the  amino  acid  was  isolated  from  the 
acid  hydrochloride  for  esterification  and  the  ester  hydrochlor- 
ide was  made  toy  decomposing  the  crude  ester  hydrochloride  accord- 

38  3 6 

ing  to  Fischer's  method  or  Foreman's  method  and  vacuum  dis- 
tilling the  amino  ester  and  preparing  the  ester  hydrochloride 
by  passing  dry  hydrochloric  acid  gas  into  the  solution  in  dry 
ether.  Identical  results  as  far  as  activity  and  purity  of  the 
diazo  ester  were  concerned  were  obtained  by  using  any  one  of 
the  methods.  The  yields  obtained  by  the  method  given,  however, 
were  much  better  and  the  method  requires  much  less  time  than  vhen 
any  of  the  other  methods  \ are  used,  mainly  due  to  the  solubili- 
ties and  the  extreme  sensitiveness  of  glutamic  acid  esters  to 
alkali.  Better  results  were  obtained  by  Foreman's  method  than 
by  Fischer's  method  for  isolating  the  free  amino  esters,  but  it 
is  quite  difficult  to  remove  all  of  the  hydrochloric  acid  by 
solid  barium  hydroxide.* 

The  use  of  sulphuric  acid  for  the  diazotazation  was  found 

3 7 j 

preferable  to  the  use  of  acetic  acid  as  used  by  Uoyes  and  Marvel 
because  of  the  difficulty  of  removing  all  of  the  acetic  acid 
from  the  ether  solution,  and  also  more  particularly  because  with 
the  use  of  acetic  acid  the  ether  solution  always  contained  some 
of  the  amino  ester,  in  some  cases  as  high  as  four  per  cent  of 
the  product.  This  necessitated  repeated  washing  with  cold  dilute 
acids  for  the  removal,  and  a consequent  production  of  a large 
amount  of  hydroxy  ester.  It  was  found  that  in  the  case  of  di- 
ethyl aiazoglutarate  a nearly  complete  purification 


' 


could  be  accomplished  with  only  a slight  loss  of  diazo 

ester  by  diluting  the  dried  ether  solution  of  the  diazo  ester 
to  a large  volume  with  anhydrous  ether,  and  adding  to  the  dilute 
solution  the  calculated  amount  of  a H ether-methyl  alcohol  solu-i 
tion  of  sodium  methylate  to  form  presumedly  the  sodium  alkoxide 
salt  of  the  hjrdroxy  ester.  Upon  the  addition  of  the  sodium 
methylate  solution  the  ether  solution  was  turned  from  a bright 
citrous  yellow  color  to  a light  reddish  brown,  but  upon  stand- 
ing a yellow  precipitate  separated,  and  the  ether  solution  was 
left  only  slightly  darker  than  the  original  color.  The  ether 
solution  was  then  filtered  and  washed  with  cold  water  as  long 
as  the  water  was  at  all  colored,  then  carefully  dried  over  cal- 
cium chloride,  the  ether  evaporated  in  vacuo  on  a water  bath 
o 

heated  to  35  , and  the  product  filtered  and  dried  by  drawing  a 
stream  of  dry  air  through  the  product  under  a pressure  of  2 mm. 
for  3 hours.  The  product  was  then  submitted  to  distillation,  I 
using  a pressure  at  least  as  low  as  0.2  mm.  If  analysis  for 

38 

diazo  nitrogen  by  the  sulphuric  acid  method  was  low  the  puri- 
fication was  repeated.  After  pure  samples  of  diazo  ester  were 
once  prepared  the  refractive  index  was  used  as  a control  test  of 
purity.  The  purification  of  the  diazo  ester  by  means  of  sodium 
ethylate  is  open  to  the  criticism  that  the  diazo  esters  them- 

39  , 40 

selves  react  with  sodium  ethylate  , however,  when  the  proper 

amount  of  the  reagent  is  added  a decided  purification  is  obtain- 
ed, as  shown  by  analysis,  density  and  refractive  index.  Moreover 
nearly  identical  results  have  been  obtained  by  repeated  fraction- 


! I ! 1 


t . ... 

- .a 


» 


. ■ ■■(.«.  1 


. - * ■ • • 


■ 


v 


> 


. 


* 


• 'r  ~ 


-16- 


al  vacuum  distillation,  but  the  amount  of  pure  diazo  dietnyl- 
glutarate  obtained  by  this  method  is  small » In  the  case  of  the 
other  diazo  esters  prepared  the  yields  of  pure  product  obtained 
by  fractional  vacuum  distillation  are  much  better* 

DERIVATIVES  OP  d -GLUTAMIC  ACID. 


This  acid  has  been  prepared  by  the  hydrolysis  of  a large 
number  cf  protein  products,  including  gliadin.  For  this  work 
it  was  prepared  by  hydrolysis  of  the  crude  flour  protein  ob- 
tained by  simply  washing  the  flour  with  water  to  remove  the 
starch. 

Preparation  of  d-Glutamic  Acid  Hydrochloride. 

24  pounds  of  hard  wheat  flour  in  the  original  muslin  sack 
are  soaked  for  30  minutes  in  a sink  filled  with  water  and  then 
vigorously  kneaded,  with  frequent  changes  of  water  until  very 
little  more  starch  passes  through  the  sack.  The  lumps  are  care 
fully  broken  up  and  the  product  kneaded  until  it  has  an  even 
rubbery  feeling  and  has  no  tendency  to  stick  to  the  fingers# 

The  rubbery  mass  is  then  removed  from  the  sack  and  kneaded  in 
the  sink  to  remove  the  remainder  of  the  starch  and  an3^  lumps 
present  carefully  broken  up. 

The  crude,  wet  gluten,  weighing  about  4500  grams,  is  then 
divided  into  two  equal  portions  and  placed  in  two  5 liter 
flasks,  and  to  each  of  the  flasks  are  then  added  2 liters  of 
concentrated  hydrochloric  acid,  the  the  mixture  heated  on  the 
water  bath  for  about  two  hours  or  until  the  purple  coloration 
has  disappeared,  and  all  has  gone  into  solution  except  black 


' 


..  . V 


• 

. 

. 

* 

-17- 


humus  matter.  The  mixture  is  then  heated  to  boiling  under  re- 
flux condenser  for  24  hours,  and  filtered  hot  either  on  a 
Buchner  funnel  fitted  with  a "filtros"  plate  or  with  three 
thicknesses  of  filter  paper.  The  humus  matter  is  then  heated 
on  the  water  bath  for  10  minutes  with  an  equal  weight  of  water 
and  again  filtered  and  washed  with  hot  water  until  filtrate  is 
only  slightly  colored.  The  combined  filtrates  and  wash  waters 
are  then  concentrated  under  diminished  pressure  to  a volume  of 
2000  cc.,  poured  into  a large  beaker,  cooled,  packed  in  ice, 
and  saturated  with  gaseous  HC1  and  allowed  to  stand  in  the  ice 
box  for  at  least  12  hours,  and  filtered  either  upon  a MfiltrosM 
plate  or  three  thicknesses  of  filter  paper,  using  strong  suc- 
tion and  pressing  the  product  down  until  no  more  liquid  runs 
through.  The  crude  glutamic  acid  hydrochloride  is  then  mixed 
with  enough  ice  cold  alcoholic  hydrochloric  acid  to  thoroughly- 
wet  it  and  again  filtered  and  washed  with  cold  alcoholic  hydro- 
chloric acid,  and  then  with  ether.  The  crude  product  is  then 
dissolved  in  the  smallest  possible  amount  of  boiling  water, 
cooled,  packed  in  ice  and  saturated  with  gaseous  HC1,  and  allow 
ed  to  stand  in  the  ice  box  for  2 hours,  and  again  filtered  and 
washed  as  before,  the  washing  with  alcoholic  hydrochloric  acid 
being  carried  out  until  the  filtrate  is  nearly  colorless.  After 
one  more  reprecipitation  and  washing  the  product  should  be  per- 
fectly white. If  it  is  not  or  the  filtrate  colored  more  than 
yellow  the  product  should  be  redissolved  and  reprecipitated, 
sometimes  boiling  the  solution  with  bone  black  and  filtering 


- 


w 


. 


- 

• 

. 


. 


. 


-18- 

before  reprecipitating  will  assist  in  the  decolorizat ion,  but 
usually  is  unnecessary.  The  filtrates  from  the  reprecipitations 
are  concentrated  under  diminished  pressure  and  the  glutamic  acid 
hydrochloride  precipitated  in  the  same  way  as  given  above,  except 
that  it  is  possible  to  evaporate  the  solutions  nearly  or  quite 
to  dryness,  particularly  in  the  later  filtrates*  The  combined 
fractions  of  the  precipitates  obtained  from  the  mother  liquors 
are  washed  with  cold  alcohol,  and  then  heated  for  l/2  hour  with 
an  equal  weight  of  absolute  alcoholic  hydrochloric  acid  to  es- 
ter ify  the  glutamic  acid  filtered  from  unesterified  matter.  Then 
the  alcohol  distilled  under  diminished  pressure  and  the  sirupy 
mass  of  ester  hydrochloride  mixed  with  ten  volumes  of  10  per  cent 
HC1,  hydrolyzed  by  heating  on  the  water  bath  for  5 hours,  the 
product  concentrated  under  diminished  pressure,  and  the  glutamic 
acid  precipitated  as  the  hydrochloride  and  washed  in  the  usual 
way.  The  product  may  be  dried  on  porous  plates  for  esterifica- 
tion. Yield  of  pure,  dry  d-glutamic  acid  hydrochloride  285-310 
grams. 

It  is  found  inadvisable  to  reconcentrate  the  original 
mother  liquors  as  the  material  is  so  viscous  that  it  is  very 
difficult  to  handle  and  moreover  very  little  additional  product 
is  obtained.  For  the  alcoholic  hydrochloric  acid  used  for  wash 
purposes  the  distillate  obtained  from  esterification  of  glutamic 
acid  was  utilized. 

In  the  hydrolysis  of  the  gluten  it  is  advisable  to  reflux 
the  mixture  for  at  least  24  hours,  for  although  nearly  as  good 


. r . 


. 


. 


• 

■ 

- 

/ 

• 

• 

• 

-19- 


a yield  may  be  obtained  by  considerably  shortening  the  time, 
the  liquid  is  so  viscous  that  the  filtration  of  the  glutamic 
acid  hydrochloride  is  very  difficult,  and  the  product  carries 
much  more  foreign  matter  which  necessitates  at  least  one  more  1 
reprecipitation  to  obtain  the  pure  product. 

It  should  be  noted  that  the  best  grades  of  hard  wheat  bread 
flours  will  give  somewhat  higher  yields  of  glutamic  acid  than 
the  cheaper,  due  to  the  higher  content  of  gliadin  in  the  gluten. 
The  difference  between  the  yields  from  the  best  and  madium 
grades  however  is  not  great. 

The  pure  product  dried  in  desiccator  over  sulphuric  acid 

o 

and  calcium  carbonate  sinters  at  202  - 205  and  melts  upon 

o 

rapid  heating  at  210  • 0.6013  g of  the  product  made  up  to  a 

o 

volume  of  10.00  cc.  with  water  gave  a rotation  of  + 1.47  in  a 

o 

1 dm.  tube  in  sodium  light  at  22  0 ; f ®<  + 24.44  . 

\ 

Analy  sis  ( Kj  e ldahl ) 

Substance  0.2542,  0.2479:  13.35  cc.,  13. 03  cc.,  0.1040  N 

H3SO4 

Calculated  for  CbHio04NC1  : N,  7.63.  Found  7.65,  7.66. 
d -Diethyl  Glutamate  Hydrochloride. 

This  ester  hydrochloride  was  prepared  by  a modification  of 

4 1 

the  usual  method  which  was  found  to  give  equally  good  results 
with  considerable  smaller  amounts  of  alcohol. 

50  g of  d-glutamic  acid  hj^drochloride  ( r c=a<'I3d  + 24.44) 
was  heated  on  the  water  bath  with  100  cc  . of  absolute  alcohol 
which  had  previously  been  saturated  with  dry  hydrogen  chloride, 
until  the  acid  all  dissolved  and  then  200  cc.  of  absolute  alco- 


. 


. 


* 

> 


» * 


< 


i 


■ 


i 


-20- 


hol  were  added  and  the  heating  continued  for  one  hour.  The  mix- 
ture was  then  concentrated  to  a thick  sirup  by  evaporation  under 
diminished  pressure,  100  cc.  of  absolute  alcohol  were  then  add- 
ed and  the  mixture  again  warmed  on  the  water  bath  for  one  hour, 
and  then  the  concentration  under  diminished  pressure  repeated. 
Upon  cooling  the  crude  ester  hydrochloride  sometimes  solidified. 
Whether  it  solidified  or  not  the  crude  product  was  dissolved  in 
an  equal  weight  of  chloroform  by  warming  on  water  bath, and  then  j 
after  cooling  about  30  cc,  of  dry  ether  was  added,  which  precip- 
itated a gummy  mass  containing  some  diethyl  glutamate  and  all 
of  the  impurities  and  colored  materials.  The  clear  solution  was; 
then  filtered  and  the  diethyl  glutamate  hydrochloride  precipi- 
tated by  adding  ether  as  fine  white  needles.  The  gummy  slightly- 
colored  mass  obtained  upon  the  addition  of  the  first  portion  of 
ether  was  purified  by  dissolving  in  chloroform  ana  reprecipitat- 
ing as  before.  Yield  47-55  grams  (72. 5 - 85  per  cent  of  the 
calculated  amount).  M.  P.  96-98°,  Por  analysis  the  product  was 
dried  in  vacuo  over  sulphuric  acid  and  calcium  carbonate. 

Analysis  (Kjeldahl) 

Substance  0,2671>  0,2871:  11. 09  cc.,  11.92  cc.,  0,1040  N 
H3SO4 

Calculated  for  C0H18NO4CI  : IT,  5.85,  Pound,  5.86,  5,86. 

Other  runs  were  made  in  which  the  glutamic  acid  was  isolat- 
ed from  the  hydrochloride  by  the  addition  of  the  calculated 
amount  of  NaOH  to  the  aqueous  solution  of  the  hydrochloride,  and 

42 

by  the  ammonia  method  , and  then  esterified  in  the  usual  manner 
The  results  were  essentially  the  same  as  by  the  method  outlined. 


* 


/ 


- 


1 


. . 

. 

. 


-21- 

Other  runs  were  also  made  in  which  the  free  ester  was  isolated 

43 

from  the  crude  ester  hydrochloride  by  Fischer's  method  , and 
vacuum  distilled  and  the  hydrochloride  prepared  by  passing  dry 
hydrogen  chloride  into  the  solution  in  dry  ether,  The  results 

i 

as  far  as  activity  of  the  diazo  ester  was  concerned  were  iden-  1 
tical,  but  the  yield  was  considerably  lower,  due  to  the  solubil- 
ity and  sensitiveness  to  alkali  of  the  glutamic  ester* 

Diazo  Diethyl  Glutarate 

50  grams  of  diethyl  glutamate  hydrochloride  is  dissolved 

in  laO  cc.  of  water  and  to  this  was  added  25  grams  of  sodium 

acetate,  and  the  mixture  surrounded  by  an  ice  salt  freezing  mix- 

o 

ture  cooled  to  -10  and  then  25  grams  of  sodium  nitrite  and  J00 
cc.  of  ether  are  added  and  the  mixture  gently  stirred  by  mechan- 
ical stirrer  in  such  a way  that  the  ether  layer  was  not  mixed 
with  the  lower  layer.  Then  during  the  course  of  1 hour  50  cc. 
of  10  per  cent  sulphuric  acid  were  added,  and  the  stirring  con- 
tinued for  2 hours.  The  ether  layer  was  then  separated  and  ! 

washed  first  with  5 25  cc.  portions  of  ice  cold  N sulphuric  acid 
then  with  saturated  sodium  bicarbonate  until  very  little  CO2 
is  evolved,  and  then  with  5 per  cent  sodium  carbonate  until  the 
sodium  carbonate  layer  was  distinctly  colored,  and  then  with 
water.  The  ether  layer  was  then  dried  first  with  calcium  chlor- 
ide containing  a little  anhydrous  barium  hydroxide,  and  finally 
over  fused  calcium  chloride.  The  ether  solution  was  then  dilut- 
ed with  anhydrous  ether  to  a volume  of  exactly  1 liter,  and 
exactly  10  cc.  of  the  ether  solution  drawn  off  by  means  of  a 
pipette  and  the  ether  evaporated  off  in  vacuum  desiccator  over 


- 


* 


- 


. 


. 


' 


- 

* i 

■ 


* 


-22- 

sulphur  ic  acid  and  sodium  hydroxide*  This  sample  was  then  anal- 
yzed for  nitrogen  by  the  sulphuric  acid  method*  The  difference 
between  the  theoretical  amount  of  nitrogen  and  that  found  was 
considered  to  be  due  to  hydroxy  ester  and  the  calculated  amount 
of  a H ether  - methyl  alcohol  solution  of  sodium  methylate  added 
to  combine  with  the  hydroxy  ester*  The  sodium  methylate  solu- 
tion was  made  by  dissolving  2 *30  grams  of  pure  sodium  in  21 
grams  of  absolute  methyl  alcohol,  and  diluting  to  100  cc.  with 
anhydrous  ether.  After  the  addition  of  the  sodium  methylate 
solution  to  the  ether  solution  of  the  diazo  ester  the  mixture 
was  allowed  to  stand  for  2 hours  and  then  filtered.  The  ether 
solution  was  then  washed  with  water  and  again  dried  over  fused 
calcium  chloride,  and  then  the  ether  evaporated  off  in  vacuo 
and  the  diazo  ester  dried  by  evacuating  the  flask  to  2.  mm.  and 
drawing  a stream  of  carefully  dried  air  through  the  product  for 
5 hours.  The  product  was  then  filtered  to  separate  a solid  pre- 
cipitate, probably  diethyl  aziglutarat e,  as  it  appears  exactly 
like  the  product  obtained  under  similar  circumstances  from  di- 

44 

ethyl  diazo succinate  . The  ether  solution  of  this  solid  pro- 
duct was  inactive*.  The  crude  diazo  ester,  which  analysis  by 
the  sulphuric  acid  method  shovted  to  be,  in  different  runs  from 
92  - 96  per  cent,  pure  was  then  vacuum  distilled,  using  a pres- 
sure at  least  as  low  as  0.2  mm.  The  purified  ester  boils  at 

92  - 93  at  0.1  mm.  The  yield  of  crude  diazo  ester  before  treat- 
ment wi.th  sodium  methylate  varied  in  different  runs  from  15  - 21 
grams.  The  yield  of  pure  vacuum  distilled  ester  amounts  to  8 — 


, 


# 1 I 


' 


■ 


. 


‘ 

y 

■ 

, • • 

■ , 


. 


- 


■ 

' 


-23- 


12  grams  when  the  distillation  goes  well*  Sometimes  however 
without  apparent  reason  decomposition  takes  place  even  with  a 
vacuum  of  *1  mm  and  if  tnis  takes  place,  even  lowering  the  pres- 
sure ts>o  .01  mm  or  less,  fails  to  yield  a pure  product  and  in 
this  case  the  distillate  contains  besides  diazo  ester,  hydroxy 
ester,  and  probably  the  unsaturated  ester  as  the  product  after 
destroying  the  diazo  ester  1 by  treatment  with  dilute  acid  decol- 
orizes permanganate  solution  but  had  nc.  action  oaoromine  in  car- 
bon tetrachloride.  Thirty  preparations  of  tnis  diazo  ester 
were  made,  using  both  sulphuric  acid  and  acetic  acid  for  the 
diazot izat ion,  and  the  only  cases  in  which  an  inactive  diazo 
ester  were  obtained  was  in  the  cases  where^good  vacuum  for  dis- 
tillation was  not  available.  Three  preparations  which  were 
distilled  tuth  ;a  presaurg:.  of  1.-3,  mm  besides  not  giving  pure 
samples  due  to  decomposition  the  products  were  inactive.  Of 
the  preparations  which  were  distilled  with  a vacuum  of  .2  mm 

or  less  the  rotation  of  the  samples  varied  from  [C^<JD+  0.37°  to 
o 

+1.68  . Three  preparations  were  made  in  which  the  analysis 
showed  to  be  9^*5  - 99  per  cent  pure  and  the  rotations  varied 
in  these  cases  f r om r<=><~^  + 1.38  to  + 1.68  and  these  prepara- 
tions as  well  as  the  others  showed  substantially  the  same  rota- 
tion in  the  pure  state  as  in  the  crude  state  (before  distilla- 
tion). Six  oth^r  preparations  were  made  which  the  analysis 

showed  to  be  99  - 98  per  cent'pure  and  these  preparations  all 

0 0 

had  rotations  of  + 0.8?  to  + 1.68  . A typical  set  of 

data  on  one  sample  are  given:- 


• 


, 


• 

■ 


- 


* •' 


-24- 


o 0 

d 1*124;  Rotation  in  a 1.  dm  tube  in  sodium  light  at  20 

+ 2.01°;  t><3  X)10  + 1.68° i 4°  1.4730. 

Substance  0,11?,  0.1088  : 15*25  cc  of  nitrogen  at  24° 
and  747  mm 

Calculated  for  CeHi+O+Hal  H,  13»08*  Found:  lJ.OO,  12*92 
(Sulphuric  acid  method) 

Substance  0.2280,  0.2047  : 0.4206  C0a,  0.1599  Ha  0 and 
O.5785  C0a,.  0.1250  KaO 

Calculated  for  C^Hi-tO^Na  : c,  50.44,  H,  6. 59*  Found:  C, 
50.55,  H,  6.86;  C,  50.40,  H,  6.78. 

Dumas  Method  1 

Substance  0.2117,  0.1989.*  25.22  cc  H fat  25°  and  744  mm., 
25.80  cc  II  at  24°  and  759  mm. 

Calculated  for  C9H14O4H2:  N,  15.08.  Found  II,  15*15>  15*21 
Large  samples  were  run  with  particular  pains  to  determine 
hydrogen. 

Substance  0.5285,  1.0015  : 0.5148  HaO,  0.5994  HaO 
Calculated  for  C9H14O4II3  : H,  6.59*  Found,  H,  6.62,  6.65*  ■ 
The  residues  from  the  sulphuric  acid  nitrogen  det erminat ioj i 
were  neutralized  with  sodium  hydroxide  and  concentrated  under 
diminished  pressure  and  made  up  to  a volume  of  5*00  cc  and  2 
cc  samples  run  on  a micro  Van  Slyke  machine. 

o 

Substance  .0447,  0,0447  : 0,01  cc  nitrogen  at  24  and  74? 
ram.,  0,015  H at  24°  and  747  mm. 

Per  cent  diethyl  glutamate  in  sample:  0,10  per  cent. 

Molecular  Weight  by  Boiling  Point  of  Ether  Method. 

0,6494  g of  diazo  ester  raised  boiling  point:  of  68.4  g of 
etner  .0910,  Mol.  wt.'220.  Repeated  experiment  and  boiling 
point  raising  was  .095°.  Mol.  Wt.  215.5.  0,9788  g of  diazo 


/ 


. 


\ 


-25- 


ester  raised  the  boiling  point  of  69*5  g of  ether  0.136  . hoi. 
wt . 2 18 . 

Calculated  for  CaHi+CUNa  : 214.15 

Samples  of  the  diazo  were  tested  for  halogen  both  by 
Beilsteiii  copper  wire  method  and  sodium  fusion  and  showed  no 
halogen. 

Treatment  of  Biethyl  Diazoglutarate  with  Dilute  Sulphuric  Acid. 

Fifteen  grams  of  the  optically  active  diaso  esterfochJ  d 
0 

+ I.80  were  added  to  100  cc.  of  20  per  cent  sulphuric  acid  and 

shaken  at  intervals  until  the  product  lost  its  yellow  color, 

the  product  was  then  extracted  with  ether,  the  ether  solution 

dried  over  anhydrous  sodium  sulphate,  the  ether  evaporated  on 

the  water  bath.  Yield  7 grams.  The  crude  product  was  filtered 

0 

and  roation  taken  in  a 1 dm.  tube  in  sodium  light  + 3*30 

The  product  was  then  vacuum  distilled  and  5 grams  of  product 

0 

boiling  at  90-95  at  0.2  mm  were  obtained. 

20°  ; 

d 1.072;  Rotation  in  a 1 dm.  tube  in  sodium  light 

o 

was  + 1.28  ; 

The  product  was  again  vacuum  distilled  and  4 grams  of  pro-  i 

o 20° 

duct  boiling  at  90.5  - 9i»5  at  0.2  mm  were  obtained,  d 40 

3° 

1.072;  hp  1.4484.  Rotation  in  a 1 dm.  tube  in  sodium  light 

O 0 r 20  0 

at  20  was  + I.29  ; + 1.07  . The  product  had  no  action 

with  a solution  of  bromine  in  carbon  tetrachloride  in  the  cold, 
but  readily  reacted  with  dilute  1 per  cent  potassium  permangan- 
ate. The  product  prepared  using  IT  sulphuric  acid  for  the 
hydrolysis  gave  identical  results/  and  the  purification  by  steam 

-J 


-26- 


distillation  gave  identical  results* 

Substance  0.1731,  0,1749;  C02,  0.3445,  0.3493*  HaO, 

0.1156,  0.1168 

Calculated  for  CeHi404l  C,  5o»10,  H,  7*57 
Calculated  for  C7H10O4!  C,  53*10;  H,  6*33 
The  analysis  reaction  with  potassium  permanganate  and  the 
decrease  in  rotation  upon  distillation  which  does  not  change 
with  further  distillation  indicates  that  the  product  is  a mix- 
ture of  diethyl  glutaconate  and  the  lactone  ester^ ethyl  tetra— 
hydro-5-keto-2-furancarboxylate  ). 


Saponification  of  the  Acid  Hydrolysis  Products  of  Diethyl  Diazo)-- 
glutarate. 

2.0  g of  the  acid  hydrolysis  products  from  the  preceding 
experiment  were  treated  with  (9.-6  ec.  of  pure  IT  alcoholic 
sodium  hydroxide  (from  sodium)  and  the  mixture  allowed  to  stand 
at  room  temperature  until  the  solution  was  neutral  to  phenol- 
phthalein. 

The  sodium  salt  starts  to  precipitate  immediately  after 

mixing.  After  the  saponification  was  completed,  the  sodium  salt 

was  filtered  off  and  washed  with  alcohol  and  then  with  ether, 

and  the  product  dried  in  desiccator  over  sulphuric  acid.  Yield 

0.9  grams  (SOper  cent  of  the  calculated  amount). 

O.585O  g of  the  sodium  salt  dissolved  in  water  and  made  up 

0 

to  5*00  cc  had  a rotation  of  + .14  in  a 1 dm  tube  in  sodium 
0330  45 

light  at  23  ;[c*<J  D + 1,20  . Pischer  and  Warburg  gives  the 

0 

rotation  of  l-sodiumo<hydroxyglutarate  as  - 8.65  , 


- 


« 

. 

■ 

. 


• 

• 

‘ t 

« 

. 

. 

, 

4 

. 

-27- 


therefore  the  salt  prepared,  is  about  13  per  cent  active*  Ihree 

bther  preparations  of  this  salt  were  made  and  the  rotation  was 

o 

always  between  + 1*0  and  + 1.20  * 

The  product  was  tested  for  amino  nitrogen  in  a micro  Van 

Slyke  machine  and  showed  no  amino  nitrogen.  The  product  gave 

no  test  for  nitrogen  after  a sodium  fusion. 

Substance  O.5833,  0.4461:  0.4329,  0.3342  ttaaSCU 

Calculated  for  CeHeOeJ3a3:  Ha,  23.97.  Found  24.14,  24.21. 

In  the  first  preparation  of  the  sodium  salt  alcoholic 

sodium  hydroxide  was  used  which  had  been  prepared  some  time 

before  from  ordinary  " C.P.M  sodium  hydroxide.  A sodium  salt 

0 

was  obtained  which  had  a specific,  rotation  of  8.36  which  com- 

4 6 

pared  well  with  the  value  given  by  Fischer  for  the  pure  active 
sodium  salt.  The  analysis  for  sodium  however  was  slightly  too 
high.  Another  preparation  from  the  same  hydroxy  ester  with 
the  pure  sodium  hydroxide  gave  the  results  shown  above.  The 
descrepency  was  evidently  due  to  the  presence  of  sodium  carbon- 
ate in  the  sample  prepared  with  the  impure  sodium  hydroxide. 

0 

As'  it  was  found  possible  to  get  rotations  as  high  as  foci]  + 40,, 
by  the  addition  of  sodium  carbonate  to  the  solution  of  the 
sodium  <=<  hy dr oxy  glutarate.  This  great  influence  on  the  rota- 
tion of  hydroxy  acids  by  foreign  salts  has  been  studied  in 

46,47,48, 40 

detail 

For  comparison  with  the  product  obtained  by  hydrolysis  of 
the  diazo  ester,  the  amino  ester  hydrochlor ide  was  diazotized 
in  such  a way  that  the  diazo  ester  was  decomposed  without  being 
isolated. 


■ ■ 


' 


- 


. 


- 

. 

* 

■ ' 

-23- 


30  g of  d-diethylglutamat e hydrochloride  was  dissolved  in 

o 

320  cc  of  N sulphuric  acid  and  solution  cooled  at  0 and  then 

to  the  solution  were  added  slowly  18  g of  sodium  nitrite  dissolv 

0 

ed  in  43  cc  of  water.  The  mixture  was  allowed  to  stand  at  0 

for  1 hour  after  the  addition  of  the  sodium  nitrite  and  then 

removed  from  the  cooling  hath, and  allowed  to  stand  at  room  tem- 

0 

perature  for  3 hours, and  finally  warmed  to  40  for  1 hour,  l’ne 

mixture  was  then  extracted  with  ether,  the  ether  solution  dried 

over  anhydrous  sodium  sulphate  and  the  ether  evaporated  on 

water  bat^h  and  the  product  vacuum  distilled,  collecting  frac- 

o 

fcion  boiling  at  90-95  at  .2  mm.  Yield  3 grams.  This  fraction 

0 

was  redistilled  collecting  fraction  boiling  at  90.5  - 91*5 

.2  mm.  Yield  5 g. 

20°  20° 

d 1.072;  nj  1 .4485;  Rotation  in  a 1 dm.  tube  in 

o o _ _23°  O 

sodium  light  at  23  +2.04  D + 1.90  . 

Substance  : 0.1966,  0.1780;  C02,  0*3917*  0.3551;  HaO,  0.134*, 
0.1189 

Calculated  for  CeHieOs  : C,  52.91,  H,  7*90.  Found:  C, 54.34 


54.40  ; H,  7*52,  7*42. 

The  product  was  saponified  as  described  in  the  preceding 
experiment.  From  3 grams  of  the  ester  1.1  gram  of  sodium  salt 
was'  obtained. 

Substance  : 0*3242  : 0.2417  HaaS04 

Calculated  for  CcHeOeNaa  : Na,  23.97.  Found:  Ma,  24.18. 

0.5672  dissolved  in'water  volume  of  solution  5. 00  cc. 

0 0 23 

Rotation  in  a 1 dm.  tube  in  sodium  light  at  23  + *21  p 

o 

1.85  . 


-29- 


Reduction  of  Diethyl  Diazoglutarate 

5 . g of  the  diazo  ester  was  dissolved  in  cc  of  ether  and 

BO 

1*9  g of  aluminium  amalgam  which  had  been  freshly  prepared 
and  washed  with  alcohol  were  added*  There  is  a vigorous  reac- 
tion with  a copious  evolution  of  ammonia*  If  the  reaction  became 
too  vigorous  the  contents  of  the  flask  were  diluted  with  ‘dry 
ether.  The  flask  was  frequently  shaken  for  four  hours  wirh  the 
occasional  addition  of  a drop  of  water,  1 cc  of  water  was  then 
added  and  the  flask  allowed  to  stand  overnight.  The  ether  sol- 
ution was  then  filtered  and  the  aluminium  hydroxide  washed  with 
ether.  The  ether  solution  was  then  evaporated  in  vacuo, the  re- 
sidue had  the  appearance  and  odor  of;  diethylglutarate.  2*7141 
g of  residue  made  up  to  5*00  cc  with  ether  had  a rotation  of 
+ 0.90°  in  a 1 dm.  tube.  To  the  residue  were  added  20  cc  of  10 
per  cent  hydrochloric  acid,  and  the  acid  solution  extracted  with 
ether,  to  remove  the  hydroxy  ester.  The  acid  solution  was  then 
heated  on  the  water  bath  for  5 hours  and  the  solution  evaporated 
nearly  to  dryness  under  diminished  pressure.  Upon  cooling  beau- 
tiful white  crystals  formed  on  walls  of  flask  which  had  the 
characteristic  taste  of  glutamic  acid.  The  crystals  were  washed 
from  the  flask  with  a little  alcohol  and  ether  added  to  complete 
the  precipitation.  Yield  from  different  preparations  .75  -1*1 
g (30  - 43  per  cent  of  the  calculated  amount).  The  product  was 
dissolved  in  water  and  reprecipitated  by  adding  alcohol  and  then 
ether. 


0.2511  g. dissolved  in  water  and  made  up  to  a volume  of  5. 00 


u'»  ,'v.' 


• v •«**.- 

J 

* 

* • V J>  • 

- 

> t • . . ■ • 

. 


V.  . 


. . ■■ 


. . , 


w 

. . . - 

. 


• % 


. , 


...  V-  * 


i.  . 


. i 


C)  " 


-30- 


o 

cc  had  a rotation  of  + .16  in  a 1 dm.  tube  in  sodium  light  at 

o 2 3 o 

23  ; + 3*20  . 

•3829  g dissolved  in  water,  volume  of  solution  3*00  cc  had 

0 0 

a rotation  of  + . 36  in  a 1 dm.  tube  in  sodium  light  at  24  ; 

24  0 

[c*do  + 3.10 . 

The  rotation  of  the  glutamic  acid  hydrochloride  from  3 other 

0 0 

runs  varied  fromcpOL  + 2.90  to  + 3. 20  • 

Substance  0.1134,  0.1214  : 5*89  cc.,  6,27  cc.  0.1040  N H2SQ4 
Calculated  for  C15H10O4NCI  : N,  7*63*  Found  7 •36,  7*32. 

Attempts  to  Prepare  Diethyl  Diazoglutarate  by  Oppe's  Method. 

Bi 

Ethyl  5 pyrrolidone  2 carbo^ylate.  Fischer  and  Bolhner 
have  prepared  this  compound  by  heating  of  diethyl  glutamate  and 
found  that  it  was  not  racemized  in  the  preparation  as  it  gives 
d-glutamic  acid  with  full  activity  upon  hydrolysis. 

15  g of  d-diethyl  glutamate  was  heated  for  20  minutes  on 

0 

an  oil  bath  to  160-170  under  a pressure  of  20  mm  with  a water 

pump,  the  flask  was  then  evacuated  with  oil  pump  and  the  product 

distilled.  The  contents  of  the  flask  nearly  all  distilled  over 

0 

at  a constant  temperature.  Boiling  point  137  - 140  .43  rmn. 

Upon  pouring  the  distillate  out  into  open  dish  the  product  crys- 
tallizes in  white  needles.  Yield  9*3  g ( 80  per  cent  of  the  cal- 
culated amount ).  After  recrystallization  from  ether  solution 

0 

by  the  addition  of  petroleum  ether  the  product  softens  at  49-50 

o 

and  melts  at  54  ♦ 

Substance  0.4511,  O.3556  : 29*01  cc.  0.09872  11  HC1( Kj elhahl 
Calculated  for  C7H11O3U  : N,  8. 91.  Found:  8.86>  8.84. 


L. 


-31- 


2.0013  % dissolved  in  water  and  made  up  to  a volume  of 

10.00  cc  gave  a rotation  of  -.49  in  a 1 dm.  tube  in  sodium 

o 

light ; -2.45  . Neither  this  product  or  the  free  acid 

made  by  heating  d-glutamic  acid  in  a similar  manner  gives  a 
nitroso  derivative  when  treated  with  hydrochloric  acid  and 
sodium  nitrite,  sodium  nitrite  in  glacial  or  50  per  cent  ace- 
tic acid  (or  in  solution  in  dry  ethe  r when  treated  with  amyl 
nitrite  and  dry  hydrogen  chloride). 

d-Dimethyl  Glutamate  Hydrochloride. 

The  crude  ester  hydrochloride  was  prepared  exactly  as  des- 
cribed for  the  diethyl  compound,  the  ester  hydrochloride  would 
not  crystallize  upon  cooling  and  the  product  would  not  crystal- 
lize when  precipitated  from  chloroform  solution  by  means  of 
ether i 

Dimethyl  «=<Diazoglutarate 

This  diazo  compound  was  prepared  from  the  crude  amino 
ester  hydrochloride  exactly  as  described  for  the  diethyl  ester 
from  3 0 g.  of  the  d-ester  hydrochloride , there  was  obtained 

f 

12  g of  the  crude  diazo  ester.  After  vacuum  distillation  there 

o 20° 

was  obtained  6 g of  product  boiling  at  86  - 87  *5  nun;  d 40 

I.I83;  njj  1.4730  at  20  ; Rotation  in  a 1 dm.  tube  at  22°  +1.04 
23  ^0 

CcK.*]  x)  + *88  • The  product  was  redistilled  and  3 g of  pro- 

0 p o° 

duct  was  obtained  boiling  at  85  - 86  ,4  mm  d—~  1.185  n-^ 

1.4753'  Rotation  in  a 1 dm.  tube  in  sodium  light  at  22  +1.06; 
22  0 9 

+ »89  . 


-32- 


Substance  : 0,2203,  0,2091  : 29.87  cc  N at  230  and  739  mm. 
28.33  cc  N at  22°  and  740  mm. 

Calculated  for  C7H10O4U2:  N,  15*07*  Found:  14.78,  14.83* 

! 

Another  preparation  of  this  diazo  ester  was  made  in  exactly 
the  same  way  except  that  a lower  pressure  was  used  in  the  dis- 
tillation, the  results  were  exactly  the  same.  Eoiling  point 
o 

82  - 83  0.2  mm. 

d-Isopropyl  Glutamate 

The  crude  ester  hydrochloride  was  prepared  in  the  same 
manner  as  described  for  the  diethyl  ester  hydrochloride,  except 
that  it  was  necessary  to  heat  the  reaction  mixture  for  about 
3 times  as  long.  The  crude  ester  hydrochloride  would  not  crys- 
tallize upon  cooling,  and  could  not  be  crystallized  from  chloro- 
form by  the  addition  of  ether,  so  the  free  ester  was  liberated 
in  the  usual  manner  and  the  product  vacuum  distilled.  From  50 

g of  glutamic  acid  'hydrochloride  22  g of  product  were  obtained, 

0 

boiling  at  115  - 117  0.15  mm.  The  product  is  a water,  white 

viscous  oil. 

20°  20° 
d -40  1.023  1.4402 

0 0 

The  rotation  in  a 1 dm.  tube  in  sodium  light  at  22  is  +5*65 

2.2.  ® 
d +5*o8  . 

Analysis  (Kjeldahl) 

Substance  O.2739,  0,2853:  11,49  cc.,  11.97  cc.,  0.1040  N 

H2SO4 

Calculated  for  C11H21HO4  : N,  6,06.  Found:  6.11,  6.10. 

d-I so propyl.  Glutamate  Hydro chloride 

ihe  ether  solution  of  the  free  ester  was  treated  with  dry 


hydrogen  chloride,  and  the  ester  hydrochloride  was  precipitat- 
ed as  a thick  viscous  oil*  The  ether  was  decanted  and  the  re- 
mainder of  the  ether  and  hydrochloric  acid  removed  in  vacuo 
over  sulphuric  acid  and  sodium  hydroxide*  The  yield  was  prac-  J 
tic ally  quantitative. 

Diisopropyl-Diazoglutarate 

The  preparation  of  this  diazo  ester  was  carried  out  ex- 
actly as  described  for  the  diethyl  ester,  using  the  ester 
hydrochloride  prepared  by  precipitation  of  the  free  ester  from 
ether  solution  by  dry  hydrogen  chloride.  From  20  grams  of  the 
ester  hydrochloride  there  was  obtained  6 grams  of  crude  pro- 
duct, the  product  was  washed  and  dried  as  described  for  the 

o 

diethyl  ester.  The  crude  product  had  a rotation  of  + 1.24  in 
a 1 dm*  tube  in  sodium  light.  The  product  was  tested  for  amino 
nitrogen  by  the  Van  Slyke  method*.  0.1084  was  warmed  with  504 
acetic  acid  until  yellow  coloration  was  destroyed,  and  then 

i 

"gave  only  0-04  cc.  of,  nitrogen  T.?han  treated  in  a micro.  Van  Slyke 
^machine.  An  attempt  to  distill  a portion  of  the  product  results! 

;in  a large  amount  of  decomposition. 

Diisopropyl  o<  Hydroxy  Glutarate. 

The  remainder  of  the  crude  diazo  ester  prepared  above  was 
shaken  with  20  per  cent  sulphuric  acid  in  the  usual  manner 
until  the  3^ellow  cloor  disappeared.  The  mixture  was  then  ex- 
tracted with  ether,  the  ether  layer  dried  over  anhydrous  sodium 
sulphate,  the  ether  evaporated  off  on  the  water  bath  and  the 


. — 


h i 


>- 


. 


■ 


V 


L 


; 


-34- 


product  distilled  with  steam.  Yield  2 grains.  The  product  is 

slightly  heavier  than  water,  n 1.4440.  1.4732  g of 

product  dissolved  in  ether  volume  of  solution  5*00  cc.,  rota- 

o o 24 

tion  in  a 1 dm  tube  in  sodium  light  at  24  + .33  ; [o<  J -p 

o 

+1.12  ♦ The  product  gave  no  test  for  amino  nitrogen  when 
tested  by  Van  Slyke  nitrous  acid  method. 


Attempt  to  Prepare  d-Di-n-butyl  Glutamate. 

Fifty  grams  of  d-glutamic  acid  hydrochloride  was  treated 
in  the  same  way  used  for  the  preparation  of  the  diisopropyl 
ester.  The  product  after  the  evaporation  of  the  excess  alcohol 
would  not  crystallize,  and  the  product  was  soluble  in  ether. 

The  crude  product  was  treated  with  potassium  carbonate  and  con- 
centrated sodium  hydroxide  in  the  usual  manner  to  obtain  the 

62 

free  amino  ester  and  the  crude  product  vacuum  distilled. 

o 

Yield  20  g B.P.  151-153  at  0.2  mm.  The  product  is  a colorless, 
very  viscous  sirupy  liquid  with  a small  amount  of  flaky  mater- 
ial floating  in  it.  The  product  was  redistilled  and  obtained 

as  a clear  oil.  The  product  would  not  crystallize  when  immers- 

20°  20 

ed  in  a bath  of  salt  and  ice.  d 1.1101;  I.4773 . 

o 32 

Rotation  in  a 1 dm.  tube  in  sodium  light  at  22°  -13*75  >£°Od 
-12.39°* 

Substance  0.8499,  0.3201:  46.58,  17.62  0,0979  N KC1 

( Kj  eldahl ) 

Calculated  for  CiaHacNO*:  B,  5*40*  Found:  N,  7*53,  7*55 
Calculated  for  CeHicOsN  : 7.56, 

The  product  did  not  react  when  treated  with  an  equivalent 

0 

amount  of  hydrochloric  acid  and  sodium  nitrite  at  0 . The 


-35- 

product  evidently  is  butyl  5 pyrrolidone  2 carboxylate.  It 
probably  was  formed  during  the  esterification  and  not  upon  dis- 
tillation as  the  crude  esterification  mixture  after  evapora- 
tion of  the  excess  alcohol  was  soluble  in  ether* 


1-Diethyl  Asparate  Hydrochloride. 

The  crude  ester  hydrochloride  was  prepared  from  1-asparag- 

sa 

in  according  to  the  method  of  Curtius  and  Lang  and  was  re- 
crystallized from  chloroform  solution  by  means  of  ether  as 
described  for  d-diethyl  glutamate.  From  30  g of  asparagin 

0 

there  was  obtained  27  g of  the  ester  hydrochloride.  M.P.  95  * 
Diethyl  c^Diazosuccinate. 

This  compound  was  prepared  from  the  recrystallized  amino 
ester  hydrochloride  according  to  the  method  of  Curtius  and 

" 6 4 

Muller  . From  27  g of  the  ester  hydrochloride  there  was  ob- 
tained after  two  distillations  7 S of  product,  boiling  at  77“ 

0 

78  at  0.1  mm. 

20° 

d 4 1.139  Rotation  in  a 1 dm.  tube  in  sodium  light 

O 0 38  o 20 

at  22  - 1.40  , L^<  - 1.23  hp  1.4620 

The  product  was  analyzed  by  the  sulphuric  acid  method. 

0 

Substance  0.2106,  0.20831  26.94,  26.53  cc  N at  24  and 
742  mm. 

Calculated  for  C8H13O4H2;  14,00.  Found;  13*93*  13*87. 

The  product  was  tested  for  amino  nitrogen  by  the  Van  Slyke 
nitrous  acid  method:  0.10  cc  of  the  diazo  ester  after  treat- 

ment with  50  per  cent  acetic  acid  until  the  yellow  color  was 
destroyed  gave  only  0.02  cc  of  nitrogen.  The  diazo  ester  was 


, 


. 

. 

* 

• 

; • 

<• 

• 

- 

-36- 


treated  with  dilute  sulphuric  acid  in  the  usual  manner.  3. 0112 

grams  of  crude  material  was  obtained,  the  product  was  diluted 

with  ether  to  a volume  of  5. 00  cc  and  the  rotation  taken  in  a 

0 024  0 

1 dm.  tube  in  sodium  light  at  24  ; cx  -0.59  “0*98  . 

cK>Amino  n-Caproic  Acid  ( Nor-leucine  ).  This  amino  acid  was 

31 »5B, 8fi 

prepared  by  the  methods  previouslu  used  and  resolved  by 

'TJ7  j 

means  of  the  brucine  salt  of  the  formal  derivative  • The  d. 

e b 

and  1 ethyl  esters  were  prepared  by  Fischer's  method  and  the 

37 

ester  hydrochlorides  prepared  by  the  method  of  Noyes  and  Marvel. 

Preparation  of  Optically  Active  Diazo  Esters  from  the  d-  and 
1-Ester  Hydrochlorides  of  cKAmino-n-Caproic  Acid* 

The  diazo  esters  from  these  esters  have  been  prepared  by 

89 

Noyes  and  Marvel  and  found  to  be  inactive,  using  substantial- 
ly the  same  method,  except  using  10  per  cent  sulphuric  acid 
instead  of  glacial  acetic  acid  and  lower  pressures  for  the  dis- 
tillation of  the  optically  active  diazo  esters  were  prepared. 

The  crude  diazo  esters  obtained  were  purified  in  the  same  manner 
as  already  described  for  diethyl  diazo  glutarate,  omitting  the 

treatment  with  sodium  methylate  however.  Using  20  grams  of  the 

23 

1-ester  hydrochloride - 7*27  ) 4*6  of  crude  diazo  ester 

were  obtained  which  after  one  vacuum  distillation  (boiling 

0 0 

point  *35  mm)  had  a specific  rotation  of  -I.89  . After 

0 

a second  distillation  2 g.  were  obtained  boiling  54-55  .35  mm: 

Tex}  -1.89,  n|°  1.4535  d f2-  .972. 

The  product  was  again  vacuum  distilled  and  1.1  g of  product 

0 0 

was  obtained  boiling  54  - 55  .35  mm. 


“37- 


30 

np  1*4543  d^o  .974 

1.0112  g dissolved  in  dry  ether.  Volume  of  solution  2,00  c. 

0 . r 24 

Rotation  in  a 1 dm  tube  in  sodium  light  at  24  - • 97>t0<J  r 

V 1 

-1.92. 

Analysis  (sulphuric  acid  method) 

Substance  O.1567,  0.2243:  24.19  cc  N at  23°  and  745  mm. 
33.50  cc.  U at  24°  and  745  mm. 

Calculated  for  C8H14O3U2  : N,  16.47*  Found:  16.27,  16.33* 

The  product  gave  no  test  for  amino  nitrogen  when  tested 

by  the  Van  Slyke  and  nitrous  acid  method  in  the  usual  way. 

When  the  product  was  hydrolysed  with  dilute  sulphuric  acid  as 

described  for  diethyl  diazo  glutarate,  the  hydrolyzed  product 

was  levorotatory  and  upon  saponification  a levorotatory  sodium 

salt  was  obtained.  The  aqueous  solution  was  acidified  and 

extracted  with  ether.  The  ether  extract  was  also  levorotatory. 

From  12  g of  the  d-ester  hydrochloride  L«=<J  ^ + 5.48 

there  was  obtained  3 grams  of  the  crude  diazo  ester,  which 

0 

after  vacuum  distillation  (B.P.  53-55  at  O.35  mm.  ) had  a 

0 

rotation  in  a 1 dm.  tube  of  +1.79  • After  a second  distilla- 

0 

tion  1.4  grams  of  product  was  obtained  boiling  at  54*53  *35 

ao  '20 

mm..  n^  1.453  g d^:  O.97;  1.1813  g were  dissolved  in 

ether,  volume  of  solution  2.00  cc.  rotation  in  a 1 dm,  tube  in 

3 3 O 

sodium  light  at  23°  1.09  . + 1.84  . 

Substance  0.1843,  0.1663*  26.82,  24.16  cc.  H at  24  and 
741  mm. 

Calculated  for  CsHi^OsHa  : N,  16.47.  Found  15*82,  15.79.  j 
The  product  was  hydrolyzed  by  dilute  sulphuric  acid  in  the 
manner  described  for  diethyl  diazoglutatat e,  the  ether  solution 


V 


-38- 

of  the  hydrolysed  product  was  dextrorotatory.  Volume  of  ether 

solution  3.00  cc  rotation  in  a 1 dm.  tube  in  sodium  light 

+ 0.22;  the  ether  was  evaporated,  the  residue  weighed  0.9211 

0 

grams  px"]  - + 1.19  * 

The  crude  hydroxy  ester  was  saponified. When  this  saponi- 
fication mixture  was  acidified  and  extracted  with  ether,  the 
ether  extract  was  also  dextrorotatory. 

Amino  Isosaproic  Acid  (Leucine).  This  amino  acid  was 

/ bo  « 

prepared  and  resolved  by  the  methods  given  in  the  literature 

ei 

The  d and  1 esters  were  prepared  by  Fischer's  method,  and 
the  ester  hydrochlorides  perpared  by  dissolving  the  free  ester 
in  ether  and  passing  in  -dry  hydrogen  chloride.  Ten  grams  of 
the  levo  leucine  ([eKl-n  -13*47°  in  20  per  cent  EC1 ) gave  9*5 
grams  of  ester  boiling  at  84-83°  at  13  mm.  °0.92. 

Rotation  in  a 1 dm  tube  in  sodium  light  at  22°  - 10.93*  ;[<*  ? D j: 
- 11.41°.  The  yield  of  the  ester  hydrochloride  was  11. 3 grams. 

Ethyl  Diazoisocaproate. 

The  ester  hydrochloride  was  diazotized  in  the  usual  man-  j 

ner.  From  11. 5 grams  of  the  hydrochloride  there  was  obtained 

0 

2.0  grams  of  the  diazo  ester  boiling  at  47-30  at  0.3  mm. 

20°  20 

a ^0  0.938  nc  1.4310;  1.8360  grams  dissolved  in  ether 

volume  of  solution  5*00  cc.  Rotation  in  a 1 dm.  tube  in  sodium 
0 

light  -.36  ;ro<]  D -1.32°^  The  product  was  redistilled  and  1.2 

0 20 

grams  were  obtained  boiling  at  49-30  at  0.30  mm.  d 0.961: 

30 

n D 1.4333* 


- 

- 

. 


-39- 


I.II38  grams  dissolved  in  ether,  volume  of  solution  3*00 

0 o 

cc;  Rotation  in  a 1 dm  tube  in  sodium  light  at  24  -0.34  ; 

0 

-1.32  . The  product  was  analyzed  by  the  sulphuric 


acid  method. 

Substance  0.1438,  0.1318,*  21.33,  22. 
7*41  mm. 

Calculated  for  CaHi-iOaila:  N,  16,47. 

15*97* 


0 

30  cc.  N at  24  and 
Round  II,  16.12, 


- - 

, 


' 


. 


-40- 


IV  SUMJiARY 

1*  Diazo  esters  were  prepared  from  the'  following  amino 
ester  hydrochlorides  by  the  Curtius  method: 

d-Diethyl  Glutamate  Hydrochloride,  d-Dimethyl  glutamate 
hydrochloride, 

1-ethyl  cxamino-n-caproate  hydrochloride,  d-ethyl  <=<amino- 
n-caproate  hydrochloride. 

l-ethylc=<aminoisocaproate  hydrochloride,  1-diethyl  asparate 

% 

hydrochloride. 

2.  The  diazo  esters  prepared  from  all  of  the  above  amino 
esters  were  found  to  be  optically  active  in  the  same  sense  as 
the  original  amino  esters. 

3*  When  the  above  diazo  esters  were  decomposed  by  dilute 
acids  the  products  are  optically  active,  in  the  same  sense 
and  to  a similar  degree. 

4.  The  products  obtained  by  the  treatment  of  diazo  esters 
with  dilute  acids  are  strongly  unsaturated,  indicating  the 
presence  of  considerable  amounts  of  the  corresponding  unsatu- 
rated esters.. 

When  the  amino  esters  are  treated  with  nitrous  acid 
under  conditions  by  which  the  diazo  esters  are  decomposed 
without  being  isolated  the  products  are  optically  active,  in 
the  same  sense  but  to  a slightly  higher  degree  than  those  ob- 
tained from  the  decomposition  of  the  purified  diazo  esters. 

6.  The  products  obtained  under  these  conditions  have  ex- 
actly the  same  physical  constants  as  those  obtained  by  the  de- 


i 


- 


•r 


-41- 


composition  oi  the  purified  diaso  ester,  except  - or  a slightly 
higher  optical  activity* 

7*  Vacuum  distillation  of  the  hydrolysis  products  of  di- 
ethyl diazoglut arate  decreases  the  activity  on  the  first  dis- 
tillation but  subsequent  distillation  causes  no  further  change 
in  activity.  Analysis  density  and  molecular  refraction  indi- 
cates that,  the  product  is  a constant  boiling  mixture  of  the 
unsaturated  ester  and  ester  lactone* 

5*  The  produce  obtained  by  steam  distillation  shows  exact- 
ly the  same  properties  and  constants* 

9*  Upon  saponification  of  the  hydrolysis  products  of  the 
diazo  esters  optically  active  sodium  salts  are  obtained  which 
rotate  in  the  same  direction  as  the  original  amino  acids* 

10*  When  tne  sodium  salts  are  acidified  and  the  mixture  ex- 
tracted with  ether  the  ether  extract  is  optically  active  in  the 
same  sense  as  the  original  amino  acids* 

11*  Upon  reduction  with  aluminium  amalgam  in  moist  ether 
solution  followed  by  acid  hydrolysis  diethylo<diazotf lutarate  7 
gives  glutamic  acid  with  a rotation  corresponding  to  approxi- 
mately thirteen  per  cent  of  the  active  acid. 

12*  Impure  diisopropyl  ok diazoglutarhte  was  prepared  which 
was  optically  active  but  it  was  largely  decomposed  upon  distil- 
lation. 

13*  Impure  ethyl  and  methyiydiazovalerates  were  prepared 
from  the  lactam  of  y amino  valeric  acid  by  Oppe*s  method*. 

14.  An  attempt  to  prepare  d-dibutyl  glutamate  by  the  usual 


* s > 

. 

, 


I 


-42- 


method  resulted  in  the  production  of  butyl  5 pyrrolidone  2 
carboxylate(p<?  -12. 39°* 

13.  It  appears  probable  that  there  are  two  forms  of  diazo 
ester,  the  one  optically  active  and  the  other  inactive,  the 
active  form  beiiig  decomposed  by  dilute  acids  with  the  formation 
of  hydroxy  esters  and  being  reducible  to  the  active  amino  acid* 
The  inactive  form  decomposing  with  the  formation  of  the  unsat- 
urated ester  and  giving  the  inactive  amino  acid  upon  reduction. 

16.  It  seems  unlikely  that  diazo  compounds  are  intermedi- 
ate products  in  the  formation  of  hydroxy  acids  when  free  amino 
acids  are  treated  with  nitrous  acid. 


* 


I 


V, 


—43“ 

V BIBLIOGRAPHY 

1*  Leverie  and  Mikeska,  J.  Biol*  Chem.,  4%,  593  (1921) 

2.  Noj^es  and  Marvel,  J.  Am.  Chem.  Soc.,  £2,  2259  (192  0) 

„ r 

3.  Schiff  and  Meissen,  C-azz.  3J.,  171  ( l88l ) : Ber.  1375(1881 

4.  Curtius,  Ber.,  l£,  2230  ( 1883 ) 

5*  Curtius,  J.  prakt.  Chem*  (2)^8,  394  ( 1888 ) 

6.  Curtius,  J.  prakt.  Chem.  38.  440  ( 1888  ) 

7.  Darapsky  and  Prabhakar,  Ber.  42,  1654  (1912  ) 

8.  Staudinger,  Ber.  49,  1884  (1916): 

Staudinger,  Becker  and  Hirzel,  Ber.  4%,  1978  (1916). 

9*  Staudinger,  Gaule  and  Siegwart,  Hel.  Chem.  Acta.,  IV  212-217 
(1921) 

10.  Staudinger,  Ber.  4$,  1884-97  (1916). 

11.  Curtius,  Ber.  1&,~  2230  (1883) 

12.  V.  Perchmann,  Ber.  22,  1889,  (1894);  28,  855  (1895  )* 

13.  Oppe,  Ber..  44,  1095  (1913  ) 

14.  Staudinger,  Eer..  2198  ( 1911 ) 

15*  Ritthausen,  J.  pr.  Chem.,  ( 1 ) 105.  239  (1868);  (2  ) 2»  354 
( 1872  ) 

16.  Pischer,  Ber.  45.,  2447  (1912) 

17%  Pischer,  Ber.  4£,  1332  (1911). 

18.  Curtius,  J.  prakt.  Chem.,  (2)2°,  477  ( 1888 ) 

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20.  Staudinger,  Ber.  4£  1884;  £9,  1978  (1916) 

21.  Ritthausen,  J.  prakt.  Chem.  ( 1 ) 2£2»  239  ( 1868  );  (2)2,  554 
( 1872  ) 

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t 

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4 


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24.  Scheibler  and  Wheeler,  Ber.  44,  2684  (1911) 

2^*  Fischer,  Ber.  2.,  2447  (1912) 

26.  Fischer  and  Weibhbold,  Ber.  .4^,  1294  (1908): 

27*  Noyes  and  Marvel,  J.  Am.  Chem.  Soc.,  ^2,  2268  (1920) 

28*  Levene,  J.  Biol.  Chem.,  .21,  348  (1919) 

29.  levene  and  Mikeska,  J , Biol.  Chem.  43.,  993  (1921) 

30.  Walden,  Ber.  28,  2772  (1899  ) 

31.  Fischer  and  Groh,  Ann.  385 . 363“372  (1911) 

32.  Taf el,  Eer.,  jL2,  2414  ( 1886  ) 

33*  Curtius,  Darapsky  and  Mttller,  Ber.,  4J*,  3140,  3161  (1908) 

34.  Hantzsch  and  Lehmann,  Eer.  ^4,  2506  (1901) 

35.  Fischer,  Ber.  34,  433  (1901) 

36.  Foreman,  Biochem.  J.,  j£9  378  (1919) 

37 » Noyes  and  Marvel,  J.  Am.  Chem.  Soc.,  42,  2270  (1920) 

38.  Curtius,  J.  prakt.  Chem.,  (2)^£,  4l8  ( 1888 ) 

39»  Curtius,  Darapsky,  and  Miller,  Ber.  4^,  3140,  3161  (1908) 

40.  Hantzsch  and  Lehmann,,  Ber.  2506  ( 1901 ) 

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42.  Abderhalden,  Zeitsch,  Physiol.  Chem.,  XL*  79  (1912) 

43.  Fischer,  Ber,  £±,  1332  (1911) 

44.  Curtius  and  Koch,  Ber.  JX*  2460  (887) 

49*  Fischer  and  Warburg,  Ber.  2447  (1912) 

46.  Schneider,  Ann.  207  Zb 7 (l88l); 

47*  Thomsen,  J.  prakt.  Chem.  (2)  33.,  149  (1887)$  Ber.  Xl»  441  ( l8o2  ) 

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. 


' 


■ 


. 


: 


-45- 


51.  Fischer  and  Bolhner,  Ber.,  14,  1332  ( 1911 ) 

52.  Fischer,  Ber.  14,  438  (1901) 

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VITA 


The  writer  was  born  near  Carlinville,  Illinois,  July  2, 
l894*  He  entered  Blackburn  College  in  the  fall  of  1912,  which 
he  attended  the  ensuing  year,.  In  the  fall  of  1913  he  entered 
the  University  of  Illinois  and  graduated  with  the  degree  of 
Bachelor  of  Science  in  Chemical  Engineering  in  1917*  He  began 
his  graduate  work  at  the  University  of  Illinois  in  June,  1917  > 
while  employed  as  full  time  assistant  in  Animal  Nutrition.  In 
June,  1918,  he  resigned  his  position  in  the  University  to  take 
up  war  work  with  the  Bureau  of  Aircraft  Production,  doing  re- 
search work  on  the  production  of  isopropyl  alcohol  and  the  oxi- 
dation of  it  to  acetone,  together  with  other  minor  problems. 

In  January,  1919>  he  reentered  the  Graduate  School  of  the  Uni- 
versity of  Illinois  and  graduated  with  the  degree  of  master  of 
Science  in  1920.  While  at  the  University  of  Illinois  he  held 
the  following  appointments : - full-time  Assistant  in  Animal 
Nutrition,  1917“l8,  Assistant  in  Chemistry,  January,  1919  to 
June,  1920,  Graduate  Assistant  in  Chemistry,  1920-21,  1921-22. 


* • * 
I 


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I 


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